PROJECT SUMMARY Homologous recombination (HR) is critical for the maintenance of genomic stability, and functions to eliminate DNA double-strand breaks and chromosomal lesions. Mutations in proteins that coordinate HR result in a variety of cancers and hereditary disorders. HR is initiated when a double-stranded DNA break is nucleolytically resected to generate single-stranded DNA (ssDNA) overhangs, which are readily coated and protected by Replication Protein A (RPA). RPA is then replaced by the RAD51 recombinase, which forms long nucleoprotein filaments to catalyze downstream strand exchange reactions to promote HR. A host of pro- and anti-HR mediator proteins control the assembly of the RAD51 nucleoprotein filament on RPA-coated ssDNA; thereby dictating the temporal and spatial establishment of HR. Mutations/defects in mediator proteins such as BRCA2 result in severe deregulation of HR causing genomic instability and associated cancers. As in many metabolic processes associated with replication, repair and remodeling of DNA, multiple proteins with DNA binding capacity function together on the same DNA template. Investigating the mechanism of how a single enzyme functions in such a multi-protein milieu is technically challenging. Our approach to address this challenge uses site-specific fluorescent versions of a protein of interest that produces a change in fluorescence upon specifically binding to ssDNA. We have utilized non-canonical amino acids (ncAA) and strain-promoted cycloaddition to generate fluorescent versions of RPA (RPAf) that accurately reports its dynamics on ssDNA, enabling us to investigate how RAD51 nucleoprotein filaments are formed on RPA coated substrates. Using this methodology, we have uncovered how each domain within RPA binds/dissociates on ssDNA and present a new paradigm for RPA function. We will capitalize on this technological advancement to determine how RPA is situated on DNA during HR and how its DNA binding domains undergo micro-dissociation during RPA-exchange (Aim 1). We will decipher how pro-HR mediator proteins displace RPA while promoting the formation of RAD51 nucleoprotein filaments during HR (Aim 2). These aims will be achieved using ncAA-based approaches to generate fluorescent proteins and a combination of ensemble and single molecule biophysical methodologies to obtain kinetic parameters for the individual steps in their mechanism of action. Beyond providing mechanistic insights on mechanism of HR, our studies will advance technology for investigating the dynamics of multi-protein DNA reactions.